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Frequently Asked Questions

MycAssay™ Aspergillus

What is the kit used for?

MycAssay™ Aspergillus is indicated for use by qualified laboratory professionals for the qualitative detection of Aspergillus spp. genomic DNA extracted from respiratory specimens from the human lower respiratory tract (e.g. bronchial samples) and serum as an aid
to diagnosis in adult patients suspected of having Aspergillus infection or allergy.

What is the Aspergillus molecular target in the assay?

18s ribosomal RNA

Can DNA extracted with other extraction kits be used successfully with the MycAssay™ Aspergillus Real-Time PCR kit?

The result is determined using clinical samples and from studies on such samples we are able to define a clinical cut off, and also sensitivity and specificity of the assay in a clinical setting. We have done this using MycXtra® so the sensitivity and specificity data we quote are only valid for the specific situation where you use clinical respiratory samples, extract Aspergillus DNA with MycXtra® and analyse with MycAssay™ Aspergillus on either the Cepheid Smartcycler, Roche LightCycler 2.0, Stratagene Mx3000 or AB 7500 platform. The same applies to serum samples which are extracted using the Roche High Pure PCR Template Preparation kit and analysed with the Roche LightCycler 2.0, the SmartCycler or the Stratagene Mx3000 series platform.

However, MycAssay™ Aspergillus works perfectly well with solutions of DNA whether obtained by MycXtra® or any other extraction system. The analytical characteristics of the kit remain the same but, because we do not know how well these other systems extract Aspergillus DNA, remove inhibitors or are free from Aspergillus contamination, we cannot give clinical specificity and sensitivity for these DNA extraction systems. In these cases the final result which states ‘positive’ or ‘negative’ should be ignored; the Ct values on the Aspergillus channel indicate the sample result. The Internal Amplification Control (IAC) and the kit Positive and Negative controls are still valid.

Can the MycAssay™ Aspergillus kit be used on any other Real-Time platforms apart from the Cepheid SmartCycler and the AB 7500?

Yes, it is also validated for use on the Roche LightCycler® 2.0 and the Stratagene Mx3000 series.  If you would like to use the kit as an RUO product, please click here for more information.

Can MycAssayTM kits be run on the Roche LightCycler 1.5?

 The MycAssayTM PCR kits have been designed, optimised and validated to work on the market leading real-time PCR platforms (Cepheid SmartCycler, Roche LightCycler 2.0, ABI 7500 and Stratagene MX3000 series instruments). Some older PCR machines are not compatible with MycAssay™ products, such as the Roche LightCycler 1.5. 
The MycAssayTM kits will not work on the Roche LightCycler 1.5, because the LightCycler 1.5 is unable to specifically detect the HEX dye used on the Internal Amplification Control (IAC) beacon.
 The LightCycler 1.5 has three channels for detecting fluorescence: 530nm, 645nm, & 710nm. In contrast, the LightCycler 2.0 has six channels 530nm, 555nm, 610nm, 640nm, 670nm & 710nm.The MycAssayTM kits require the use of the 530nm channel to detect FAM dye for the target and the 555nm (referred to as 560nm in the IFU) channel to detect the HEX dye for the Internal Amplification Control (IAC). Therefore, due to limitations in the number of channels the MycAssayTM kit cannot be applied to the LightCycler 1.5 but can run successfully on the LightCycler 2.0.

 How long does the assay take to perform?

Less than 4 hours from sample receipt to result:

Fungal DNA extraction from clinical sample ~2 hours
Real-Time PCR set-up 15 minutes
Real-Time PCR run 1.5 hours
Results analysis 15 minutes

 

What are the analytical characteristics of the assay?

The Limit of Blank (LoB) was determined analytically using samples known not to contain Aspergillus DNA:

  • Cepheid SmartCycler = Ct 38.0
  • AB 7500 = Ct 37.0

The Limit of Detection (LoD) was determined analytically using the AF293 strain of Aspergillus fumigatus of known DNA concentration and is defined as the concentration at which 95% of the time the Ct value is < LoB:

  • LoD = <50 copies of the 18S target sequence

It is known there are 37 copies of the target within the genome and thus 50 target copies represents approximately 1.3 genomes

Are there any substances which may inhibit the assay PCR?

We tested 17 different substances, the majority of which were antifungal drugs which may be present in this patient population, at clinically relevant concentrations. None of the 17 substances we tested inhibited the assay. A full list of these substances tested can be found in the kit Instructions for Use.

How did you set the clinical cut-off?

The clinical cut off at a Ct value of 36.0 was established on the Cepheid SmartCycler following review of a dataset of samples sourced from different sites and different patient populations. Different cut-offs were evaluated for the probability of differentiating between disease and non disease state (see Figure 1.)

Figure 1: The cut off that gave the best differentiation, based upon the dataset available to us, was 36.0 (Cepheid SmartCycler).

This cut-off was transferred analytically to the AB 7500 platform using a template with an Aspergillus concentration that had been shown to yield ≥ 95% positive results at the clinical cut-off on the SmartCycler. A Ct value of 33.5 was determined, which was then confirmed using templates of 3 different concentrations.

What does a Ct between the clinical cut-off and the LoB represent? How should this be interpreted?

A low level of Aspergillus has been detected. Low levels of Aspergillus DNA can be detected for a number of reasons and
may represent a transient signal in normal healthy individuals, contamination during fungal DNA extraction or in setting up
the MycAssay™ Aspergillus assay, or colonisation/disease.

What are the sensitivity and specificity values?

Respiratory samples (BAL) that had been collected from 2 hospitals, extracted using the MycXtra® kit, and stored were used to evaluate the performance of the MycAssay™ Aspergillus kit with clinical samples on the Cepheid SmartCycler. Comparisons were made to both clinical diagnosis and culture:

MycAssay™ v Clinical Diagnosis
  Clinical positive Clinical negative  
MycAssay™ positive 31 1 PPV 0.97
MycAssay™ negative 2 10 NPV 0.83
  Sensitivity 0.94 Specificity 0.91  

 

MycAssay™ v Aspergillus Culture
  Clinical positive Clinical negative  
MycAssay™ positive 29 3* PPV 0.91
MycAssay™ negative 2 10 NPV 0.83
  Sensitivity 0.94 Specificity 0.77  

 *The MycAssay™ Aspergillus kit also amplifies Penicillium spp. as it is identical to Aspergillus at the 18S target sequence. Two of these three samples grew Penicillium spp. The clinical management of both infections is identical.

Can you extract fungal DNA from other sample types e.g. blood, tissue and use it with this assay?

The assay works with any purified DNA in a research setting (RUO). However, Myconostica are working on a clinically validated protocol and has studies underway with two external sites. We expect a CE marked protocol to be available in the first half of 2010.

What are the improvements of the MycAssay™ Aspergillus kit compared to the FXG™: RESP (Asp +) kit?

  • We have re-designed the primers and Molecular Beacons and have re-formulated the assay to allow the addition of more DNA template to the Real-Time PCR, both of which have resulted in an increase in the sensitivity of the assay.
  • We have changed the fluorophores; we no longer use ATTO 647N (far red) but use HEX thus increasing the number of available platforms the assay will run successfully on.
  • The IAC has been re-optimised to be more robust.
  • Aspergillus and Pneumocystis detection have been separated in response to customer feedback.
  • MycAssay™ family means the same PCR volumes and parameters are used between the different assays, making them more user-friendly.

Can I re-freeze and re-use reagents?

We have no stability data to support this at present but a project is underway to determine the effect of freeze - thaw cycles on assay reagents. Contamination is a risk if you attempt to reuse reagents at a later date.

How often is Aspergillus found in normal lungs?

Aspergillus is an airborne organism, ubiquitous in the atmosphere; the pathogenic Aspergilli are found in air, on surfaces, soil and compost the world over. Exposure to Aspergillus spores in the air is inevitable. Most aerobiology studies have been done in Europe: the usual concentration of conidia (spores) in outdoor air is 2 - 30 conidia/m3 air. Most studies do not show a seasonal variation in airborne Aspergillus counts. However, some studies have shown an increase in the winter in temperate climates in the Northern Hemisphere. The Aspergilli comprise from 0.1 – 22.0 % of the total air flora outside. A. flavus is typically the most frequent Asperrgillus sp. found in air inside and outside; A. fumigatus comprises from 4.0 – 41.0% of the total. A fumigatus is found frequently both indoors and outside, but is not always the most common Aspergillus species indoors. Soil isolation rates increase towards the equator. Aspergilli may be found in water, especially that from surface reservoirs. Aspergilli may form biofilms in pipework, and provide a possible source of infection. It is not known how important this in terms of infection.

Most fungal exposures are of limited extent. Studies in normal lungs show that most people have culturable fungi detectable in airways and the lungs (Lass-Florl et al 1999)*, although standard fungal cultures of respiratory secretions are typically negative. Aspergillus conidia appear to be immunologically unreactive, until they start to swell and germinate. Because culture systems and DNA extraction for fungal DNA is not 100% efficient, extremely low concentrations of Aspergillus spores may not be detected, especially in very small samples.

*Pulmonary Aspergillus colonization in humans and its impact on management of critically ill patients. Br J Haematol 1999 Mar; 104 (4):745-7

For any further questions, please contact mycotech@myconostica.co.uk